For the past several years, cancer has been the leading cause of death in Japan. In addition to surgery, radiotherapy and chemotherapy, immunotherapy is the fourth option for cancer therapy. An in vivo system for immune response is utilized in immunotherapy. The immune response is elicited and controlled by the interaction amongst B lymphocytes, T lymphocytes, antibodies and antigen presenting cells (APC). First, an exogenous antigen is processed in the APC, and then it is presented in a form bound to the major histocompatibility complex (MHC) class 1 or class 2 to helper T cells. Upon the recognition by helper T cells of this exogenous antigen, T cells are activated and cytokines are secreted. The secreted cytokines help differentiation of antigen-stimulated B cells into antibody-forming cells and at the same time promote the differentiation of killer T cells. Finally, cells presenting antigens are eliminated by the secreted antibodies and by the activated killer T cells. This is how cellular and humoral responses operate in order to eliminate exogenous antigens.
The elimination process of antigen expressing cells by T cells can be broadly classified in three groups: 1) humoral immunity (activated helper T cell stimulates proliferation/differentiation of specific B cell clones, antibodies are produced and the antibodies recognize and eliminate antigens); 2) specific cellular immunity (activated helper T cells induce cytotoxic T cells (CTL) that react on specific antigens and the CTL directly reacts to the target); and 3) non-specific cellular immunity (activated helper T cells induce non-specific natural killer cells, activated macrophages, etc. and these cells function to eliminate antigens). As described above, T cells play a central role for recognizing target antigens to elicit immune response.
Regarding tumor rejection, antitumor immune responses in host cells have been known to be induced by appropriate immunization using syngeneic or self-derived tumor cells or fractions thereof (L. Gross, Cancer Res. 3: 326-333 (1943); E. J. Foley, Cancer Res. 13: 835-837 (1953); R. T. Prehn and J. M. Main, J. Natl. Cancer Inst. 18: 769-778 (1957); G. Klein et al., Cancer Res. 20: 1561-1572 (1980); L. Old et al., Ann. N.Y. Acad. Sci. 101: 80-106 (1962); A. Globerson and M. Feldman, J. Natl. Cancer Inst. 32: 1229-1243 (1964)). The role of CD8+ and CD4+ T cells in these tumor systems has been of enormous interest (R. J. North, Adv. Immunol. 35: 89-155 (1984); P. D. Greenberg, Adv. Immunol. 49: 281-355 (1991); D. M. Pardoll and S. L. Topalian, Curr. Opin. Immunol. 10: 588-594 (1998)). CD8+ T cells from specifically immunized mice are reported to be capable of destroying tumor target cells in vitro (H. Wagner et al., Adv. Cancer Res. 31: 77-124 (1980)). Furthermore, it has been reported that adoptive transfer of CD8+ T cells from immunized donors confers resistance to tumor transplants to naive mice (R. J. North, Adv. Immunol. 35: 89-155 (1984); P. D. Greenberg, Adv. Immunol. 49: 281-355 (1991); C. J. M. Melief, Adv. Cancer Res. 58: 143-175 (1992)). In addition, anti-CD8+ antibodies are known to abolish resistance to tumor transplantation in preimmunized mice (E. Nakayama and A. Uenaka, J. Exp. Med. 161: 345-355 (1985); X. G. Gu et al., Cancer Res., 58: 3385-3390 (1998); Y. Noguchi et al., Proc. Natl. Acad. Sci. USA 92: 2219-2223 (1994)). Over the past decade, MHC class I binding peptides derived from tumor cells of mice and human that are recognized by CD8+ T cells have been reported (T. Boon et al., Annu. Rev. Immunol. 12: 337-368 (1994); S. A. Rosenberg, Immunity 10: 281-287 (1999)).
Two forms exist for tumor antigens (target molecules on tumor cells). These are: (1) tumor peptide presented by MHC class I molecules, the target molecule of CD8+ CTL that is the leading character of cellular immunity; and (2) the target molecule of humoral immunity (antibody) that is expressed on the cell membrane of tumors is called tumor-associated antigen. Since a human tumor antigen recognized by a T cell has been defined at the genetic level, various human tumor rejection antigens have been discovered. Vaccination therapy is defined as a specific immunotherapy that uses a tumor rejection antigen and evident antitumor effect has been confirmed for the therapy. Furthermore, potentiation of immunotherapeutic effect by the combined use of cytokines and dendritic cells pulsed with antigenic peptides, or introduced with antigen genes has been attempted. Moreover, recently, DNA vaccines have been tested in the art.
A number of approaches to augment the helper action of CD4+ T cells have been attempted (D. Pardoll and S. L. Topalian, Curr. Opin. Immunol. 10: 588-594 (1998); R. F. Wang, Trends Immunol. 5: 269-276 (2001)). Earlier methods fall into one of three categories. One method involves modification of immunizing antigens itself. For example, haptenizing the antigen (Y. Mizushima et al., J. Natl. Cancer Inst. 74: 1269-1273 (1985)), linking heterologous immunogenic peptides directly onto the antigen (R. W. Chesnut et al., Vaccine Design, eds. M. F. Powell and M. J. Newman (Plenum, New York) 847-874 (1995); J. Rice et al., J. Immunol. 167: 1558-1565 (2001)), etc. The second is co-immunization with tumor antigens and molecules with strong helper determinants (R. Romieu et al., J. Immunol. 161: 5133-5137 (1998); N. Casares et al., Eur. J. Immunol. 31: 1780-1789 (2001)), such as viral vectors encoding tumor antigens (M. Wang et al., J. Immunol. 154: 4685-4692 (1995)). The third method utilizes molecular signals such as CD40 ligand (J. P. Ridge et al., Nature (London) 393: 474-478 (1998); S. R. M. Bennett et al., Nature (London) 393: 478-480 (1998); S. P. Schoenberg et al., Nature (London) 393: 480-483 (1998)) and other stimulatory/co-stimulatory signals (A. Porgador et al., J. Exp. Med. 188: 1075-1082 (1998)) involved in the helper function of CD4+ T cells and in modulating the interaction of APCs with CD4+ T cells. The discovery of such signals appears to provide methods to augment the response of CD8+ T cells.
Antibodies have been generally relegated to a minor role in antitumor effector functions. However, tumor antigens like NY-ESO-1 elicits a strong integrated immune response involving both cellular and humoral immunities (Y.-T. Chen et al., Proc. Natl. Acad. Sci. USA 94: 1914-1918 (1997); E. Jager et al., J. Exp. Med. 187: 625-630 (2000); E. Jager et al., Proc. Natl. Acad. Sci. USA 97: 12198-12203 (2000)). Recently, M. Pfreundschuh and his colleagues developed a method called SEREX (Y.-T. Chen et al., Proc. Natl. Acad. Sci. USA 94: 1914-1918 (1997)) to identify tumor-associated antigens that may serve as vaccines against tumors. This method involves screening of cDNA expression libraries of human tumors with human sera. More than 1800 kinds of genes identified by SEREX are registered in the SEREX database on the internet (www.licr.org/SEREX.html).
However, there are still unsolved problems. Some of the problems include the kind of adjuvant or APC to be used for effectively inducing tumor-specific immunity with the identified antigenic peptides/DNAs to finally achieve complete recovery from tumor; or dealing with the escape of tumors from the immune system.
Helper T cells are often reported to be necessary for quantitative/qualitative amplification of CTL. However, the characteristics of antigen molecules recognized by these T cells and their functional impact on antitumor immune responses are still largely unknown (P. D. Greenberg, Adv. Immunol. 49: 281-355 (1991); D. M. Pardoll and S. L. Topalian, Curr. Opin. Immunol. 10: 588-594 (1998); S. R. Bennett et al., J. Exp. Med. 186: 65-70 (1997); R. F. Wang, Trends Immunol. 5: 269-276 (2001); C. Fayolle et al., J. Immunol. 147: 4069-4073 (1991); M. Shiral et al., J. Immunol. 152: 1549-1556 (1994); K. Hung et al., J. Exp. Med. 188: 2357-2368 (1998); F. Ossendorp et al., J. Exp. Med. 187: 693-702 (1998); Y. Shen and S. Fujimoto, Cancer Res. 56: 5005-5011 (1996); T. Nishimura et al., J. Exp. Med. 190: 617-627 (1999); D. R. Surman et al., J. Immunol. 164: 562-565 (2000); A. Franco et al., Nat. Immunol. 1: 145-150 (2000); C. N. Baxevanis et al., J. Immunol. 164: 3902-3912 (2000); F. Fallarino et al., J. Immunol. 165: 5495-5501 (2000); A. L. Marzo et al., Cancer Res. 59: 1071-3390 (1999); A. L. Marzo et al., J. Immunol. 165: 6047-6055 (2000)). The current hypothesis for serial intercellular interaction amongst helper T cells, CTLs and APCs points to the possibility that helper T cells related to antitumor immune response can recognize diverse antigens of a wide range (J. P. Ridge et al., Nature 393: 474-478 (1998); S. R. M. Bennett et al., Nature 393: 478-480 (1998); S. P. Schoenberger et al., Nature 393: 480-483 (1998); Z. Lu et al., J. Exp. Med. 191: 541-550 (2000)).
Great progress is being made in the analysis of humoral immune response in human and murine tumors by the above-described SEREX method (Y.-T. Chen et al., Proc. Natl. Acad. Sci. USA 94: 1914-1918 (1997); E. Jager et al., J. Exp. Med. 187: 625-630 (2000); E. Jager et al., Proc. Natl. Acad. Sci. USA 97: 12198-12203 (2000); U. Sahin et al., Proc. Natl. Acad. Sci. USA 92: 11810-11813 (1995); L. J. Old and Y.-T. Chen, J. Exp. Med. 187: 1163-1167 (1998); Y.-T. Chen, “Principle and Practice of the Biologic Therapy of Cancer”, ed. S. A. Rosenberg (Lippincott Williams & Wilkins, Philadelphia) 557-570 (2000); T. Ono et al., Int. J. Cancer 88: 845-851 (2000)). Complete sequence determination of SEREX-defined genes show that many of them share the same sequence with the wild-type sequence, i.e., there are no amino acid substitutions included (L. J. Old and Y.-T. Chen, J. Exp. Med. 187: 1163-1167 (1998); Y.-T. Chen, “Principle and Practice of the Biologic Therapy of Cancer”, ed. S. A. Rosenberg (Lippincott Williams & Wilkins, Philadelphia) 557-570 (2000)). Therefore, these molecules do not exhibit immunogenicity due to mutations. Furthermore, although some SEREX antigens show restricted tumor expression in normal tissues (e.g., cancer/testis antigens, melanocyte differentiation antigens, etc.), most of the SEREX-defined antigens are ubiquitously expressed. However, high-titered antibodies to these wild-type molecules are present more in serum samples of associated and non-associated cancer patients compared to normal healthy subjects. The current hypothesis is that amplified expression of these tumor products serves as the immunogenic stimulus for eliciting humoral immunity. Since all of these molecules are detected by antibodies of the IgG class, these wild-type molecules imply recognition by CD4+ helper T cells. With regard to the above information, the present inventor examined in the present invention whether tumor-specific CD8+ CTL can be amplified by activating CD4+ helper T cells via immunogenic wild-type molecules of tumor cells. Namely, examined the involvement of the molecules in antitumor immune response.
DNA vaccines are demonstrated to induce both humoral and cellular immune responses upon intramuscular administration of naked DNA. The precise mechanism of induction of immune response by DNA vaccines is obscure (see, Pardoll et al., Immunity 3: 165-169 (1995)). However, its effectiveness is indicated by the induction of humoral and cellular immunities. This result indicates the expression of naked DNA following administration of a DNA vaccine, and that peptide products of the naked DNA are presented as antigens with both the MHC class I and class II proteins.
A T cell receptor on CTL recognizes an exogenous peptide derived from virus, bacteria, etc., bound to MHC class I and/or class II molecules as an antigen. Then, reactions such as production of various lymophokines and cell proliferation are known to be promoted to finally kill cells infected with the virus, bacteria, etc. Irrespective of their location in the original pathogen, these antigenic peptides are processed fragments that were intracellularly imported into APC or other cells. Known methods for artificial generation of CTL response include those using replication vectors that produce protein antigens in cells (J. R. Bennink and J. W. Yewdell, Curr. Top. Microbiol. Immunol. 163: 153 (1990); C. K. Stover et al., Nature 351: 456 (1991); A. Aldovini and R. A. Young, Nature 351: 479 (1991); R. Schfer et al., J. Immunol. 149: 53 (1992); C. S. Hahn et al., Proc. Natl. Acad. Sci. USA 89: 2679 (1992)) and methods wherein peptides are introduced into the cytosol (F. R. Carbone and M. J. Bevan, J. Exp. Med. 169: 603 (1989); K. Deres et al., Nature 342: 561 (1989); H. Takahashi et al., Nature 344: 873 (1990); D. S. Collins et al., J. Immunol. 148: 3336 (1992); M. J. Newman et al., J. Immunol. 148: 2357 (1992)).
Furthermore, a method for inoculating a vertebrate with naked polynucleotide as a vaccine has been discussed (WO90/11092 (Oct. 4, 1990)). Calcium chloride-precipitated DNA is known to be expressed via intravenous or intramuscular administration (N. Benvenisty and L. Reshef, Proc. Natl. Acad. Sci. USA 83: 9551-9555 (1986)). Moreover, in mice it was shown that DNA expression vector is incorporated into myocytes and expressed in the cell upon intramuscular injection of the vector (J. A. Wolff et al., Science 247: 1465 (1990); G. Ascadi et al., Nature 352: 815 (1991)). According to this method, the vector was sustained as an episome and did not replicate. However, permanent expression of the vector was observed following injection into the skeletal muscle of rat, fish and primate, as well as cardiac muscle of rat (H. Lin et al., Circulation 82: 2217 (1990); R. N. Kitsis et al., Proc. Natl. Acad. Sci. USA 88: 4138 (1991); E. Hansen et al., FEBS Lett. 290: 73 (1991); S. Jiao et al., Hum. Gene Therapy 3: 21 (1992); J. A. Wolff et al., Human Mol. Genet. 1: 363 (1992)). It was further reported that presentation of epitopes by B7 and MHC on the surface of APC play similar roles in the activation of CTL during tumor elimination (Edington, Biotechnology 11: 1117-1119 (1993)). When a MHC molecule on the surface of APC presents an epitope to a T cell receptor, a B7 expressed on the surface of the same APC binds to CTLA-4 or CD28 and functions as the second signal. As a result, CD4+ helper T cells that emit signals for increasing APC destroying CD8+ T cells rapidly proliferate.
For immunization with DNAs, the DNAs do not necessary have to be administered intramuscularly. For example, Tang et al. demonstrate that anti-bovine growth hormone (BGH) antibodies are produced in mice following administration of BGH-coated gold particles into the skin (Tang et al., Nature 356: 152-154 (1992)). Apart from skin, it is reported that muscular, adipose and mammary gland tissues of live animals can be transfected with DNAs (Furth et al., Analytical Biochemistry 205: 365-368 (1992)). Various methods for introducing nucleic acids are also reviewed (T. Friedman, Science 244: 1275-1281 (1989)). WO93/17706 describes a method of vaccine inoculation of an animal against a virus that comprises the steps of coating a carrier particle with a gene construct, and then administering the coated particle into a cell of the animal. Furthermore, DNA immunization against herpes virus (Cox et al., J. Virol. 67: 5664-5667 (1993)) has been reported. In addition, DNA vaccines and methods for producing and administering them are also described in U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,589,466, and WO94/16737.